Human dynein–dynactin is a fast processive motor in living cells
- Biology Department, University of Massachusetts, United States
- Molecular and Cellular Biology Graduate Program, University of Massachusetts, United States
Figures
Figure 1 with 5 supplements
Dynein tip-tracks on polymerizing microtubules (MTs) and walks processively at high velocities in interphase HeLa cells.
(A) Representative spinning disc confocal time-lapse of a DHC-EGFP-expressing HeLa cell showing robust tip-tracking. Zoomed views of the numbered boxed regions are shown to the right with kymographs of the tip-tracking events highlighted with yellow arrows in the zoomed panels. (B) Representative total internal reflection fluorescence microscopy (TIRFM) time-lapse of a DHC-EGFP-expressing HeLa cell showing the tip-tracking population. Zoomed views of the numbered boxed regions are shown with kymographs of the tip-tracking events highlighted with yellow arrows. (C) Still frame from a representative TIRFM time-lapse of a DHC-EGFP-expressing HeLa cell treated with SiR-Tubulin. In the merge image, DHC is green and Sir-Tubulin labeled MTs are magenta. (D) Still frame from a representative high temporal resolution (5 fps) TIRFM time-lapse of a DHC-EGFP-expressing HeLa cell treated with SiR-Tubulin. Boxed region is shown as a zoomed inset in the lower panel with the track of a motile DHC puncta highlighted in green. (E) Representative kymographs of motile puncta spanning the range of measured velocities (Vel) and run lengths (RL). (F) Distribution of DHC velocities (n = 100 puncta). (G) Distribution of DHC run lengths (n = 81 puncta). (H) Distribution of DHC run times (n = 81 puncta). Scale bars, 10 μm (A–D); 1 µm (all insets), 10 µm (horizontal); 1 min (vertical) in the kymographs in A and B; and 1 µm (horizontal); 1 s (vertical) in E. Displayed times are min:s.
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Figure 1—source data 1
Excel spreadsheet containing the underlying data and numerical values for plots in Figure 1.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Characterization of DHC-EGFP CRISPR knock-in cells.
(A) Repair cassette for generating the DHC-EGFP CRISPR-engineered HeLa cell line with sequencing trace showing its integration at the dynein heavy chain (DYNC1H1) gene locus. (B) Montage of representative fields of view of the CRISPR-engineered DHC-EGFP cell line used in this study. Expression levels are consistent across the population and do not exhibit significant cell-to-cell variation. Scale bar, 20 μm.
Figure 1—video 1
Spinning disk confocal time-lapse of a DHC-EGFP CRISPR-engineered interphase HeLa cell.
Zoomed views of the boxed regions in the upper left panel are shown in the right panels. Displayed times are min:s. Scale bars, 10 μm (upper left panel) and 1 µm (right panels). Relates to Figure 1A.
Figure 1—video 2
Total internal reflection fluorescence microscopy (TIRFM) time-lapse of a DHC-EGFP CRISPR-engineered interphase HeLa cell.
Displayed times are min:s. Scale bar, 10 μm. Relates to Figure 1B.
Figure 1—video 3
Total internal reflection fluorescence microscopy (TIRFM) time-lapse of a DHC-EGFP (green) CRISPR-engineered interphase HeLa cell treated with SiR-Tubulin (magenta).
Displayed times are min:s. Scale bar, 10 μm. Relates to Figure 1C.
Figure 1—video 4
High temporal-resolution (5 frames per second) total internal reflection fluorescence microscopy (TIRFM) time-lapse of a DHC-EGFP CRISPR-engineered interphase HeLa cell treated with SiR-Tubulin.
The boxed regions highlight motile DHC puncta. Displayed times are s:ms. Scale bar, 1 μm. Relates to Figure 1D.
Figure 2 with 5 supplements
The dynactin complex component p50 tip-tracks on polymerizing microtubules (MTs) and walks processively at high velocities in interphase HeLa cells.
(A) Still frames from a representative spinning disc confocal time-lapse of a p50-EGFP-expressing HeLa cell. A zoomed view of the boxed region is shown with a kymograph of the tip-tracking event highlighted with the yellow arrow. (B) Still frames from a representative total internal reflection fluorescence microscopy (TIRFM) time-lapse of a p50-EGFP-expressing HeLa cell showing the tip-tracking population. A zoomed view of the boxed region is shown with a kymograph of the tip-tracking event highlighted by the yellow arrow. (C) Still frame from a representative TIRFM time-lapse of a p50-EGFP-expressing HeLa cell treated with SiR-Tubulin. In the merge image, p50 is green and Sir-Tubulin labeled MTs are magenta. (D) Representative kymographs of motile p50 puncta spanning the range of measured velocities (Vel) and run lengths (RL). (E) Distribution of p50 velocities (n = 44 puncta). (F) Distribution of p50 run lengths (n = 41 puncta). (G) Distribution of p50 run times (n = 41 puncta). Scale bars, 10 μm (A–C); 1 µm (all insets), 5 µm (horizontal); 1 min (vertical) in the kymographs in A and B; and 1 µm (horizontal); 1 s (vertical) in D. Displayed times are min:s.
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Figure 2—source data 1
Excel spreadsheet containing the underlying data and numerical values for plots in Figure 2.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Comparison of the p50-EGFP and DHC-EGFP CRISPR knock-in cells.
(A) Montage of representative fields of view of the CRISPR-engineered p50-EGFP clone in comparison to the montage of DHC-EGFP fields from Figure 1—figure supplement 1B. For the purpose of comparison, the two cell lines were visualized with identical imaging parameters on the same day and displayed identically in the figure. (B) Scatter plots of background-corrected fluorescence intensities of cytoplasmic pools of DHC-EGFP (n = 31 cells) and p50-EGFP (n = 13 cells) from (A). (C) Montage of fields of view p50-EGFP clonal cells visualized with longer exposure times (500 ms) than in (A). Error bars are mean values ± standard deviations. Scale bars, 20 μm.
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Figure 2—figure supplement 1—source data 1
Excel spreadsheet containing the underlying data and numerical values for plots in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig2-figsupp1-data1-v1.xlsx
Figure 2—video 1
Spinning disk confocal time-lapse of a p50-EGFP CRISPR-engineered interphase HeLa cell.
Displayed times are min:s. Scale bar, 10 μm. Relates to Figure 2A.
Figure 2—video 2
Total internal reflection fluorescence microscopy (TIRFM) time-lapse of a p50-EGFP CRISPR-engineered interphase HeLa cell.
Displayed times are min:s. Scale bar, 10 μm. Relates to Figure 2B.
Figure 2—video 3
Total internal reflection fluorescence microscopy (TIRFM) time-lapse of a p50-EGFP (green) CRISPR-engineered interphase HeLa cell treated with SiR-Tubulin (magenta).
Displayed times are min:s. Scale bar, 10 μm. Relates to Figure 2C.
Figure 2—video 4
Total internal reflection fluorescence microscopy (TIRFM) time-lapse of a p50-EGFP (green) CRISPR-engineered interphase HeLa cell treated with SiR-Tubulin (magenta).
Displayed times are min:s. Scale bar, 10 μm.
Figure 3 with 2 supplements
Motile DHC and p50 puncta exhibit identical motility parameters but different fluorescent intensities.
Scatter plots of (A) velocities (DHC, n = 100; p50, n = 44), (B) run lengths (DHC, n = 81; p50, n = 41), and (C) run times (DHC, n = 81; p50, n = 41). Distributions of the background-corrected fluorescence intensities of motile puncta of (D) kinesin-1-EGFP transiently expressed in HeLa cells, (E) DHC-EGFP, and (F) p50-EGFP. The dashed line in each histogram denotes the mean value of the kinesin-1-EGFP dataset. (G) Scatter plots of the kinesin-1, DHC, and p50 fluorescence intensities (kinesin-1, n = 90 puncta; DHC, n = 84 puncta; p50, n = 74 puncta). (H) PCR of genomic DNA from the DHC-EGP clone used in this study using PCR primers flanking the integration site of the repair cassette. The upper band was extracted and subjected to sequencing, the results of which are shown in Figure 1—figure supplement 1A. (I) Western blot for p50 of cell lysates from the parental HeLa cell line and the p50-EGFP clone used in this study. The tagged p50 runs ~30 kDa larger than the untagged p50 and is expressed at ~5- to 6-fold lower levels than the endogenous p50. Error bars are mean values ± standard deviations. The reported p-values were determined by a randomization method: n.s. is not significant (p > 0.05).
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Figure 3—source data 1
PowerPoint file containing original image of agarose gel for Figure 3H, indicating the relevant PCR fragments.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig3-data1-v1.pptx
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Figure 3—source data 2
Original file of agarose gel image in Figure 3H.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig3-data2-v1.zip
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Figure 3—source data 3
PowerPoint file containing original membrane and western blots for Figure 3I, indicating the relevant bands and cell line lysates.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig3-data3-v1.pptx
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Figure 3—source data 4
Original files for western blot in Figure 3I.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig3-data4-v1.zip
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Figure 3—source data 5
Excel spreadsheet containing the underlying processed data and numerical values for plots in Figure 3.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig3-data5-v1.xlsx
Figure 3—figure supplement 1
Motility parameters of the transiently expressed Kinesin-1-EGFP in HeLa cells.
(A) Representative kymographs of motile kinesin-1-EGFP puncta from transiently transfected HeLa cells. (B) Distribution of kinesin-1 velocities (mean ± SEM; n = 76 puncta). (C) Distribution of kinesin-1 run lengths (mean ± SEM; n = 60 puncta). Kinesin-1 expressed in HeLa cells exhibited a slower mean velocity and shorter mean run length than the HeLa dynein–dynactin. Scale bars, 1 µm (horizontal); 1 s (vertical).
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Figure 3—figure supplement 1—source data 1
Excel spreadsheet containing the underlying data and numerical values for plots in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
Fluorescence intensity comparisons to Kinesin-1-EGFP transiently expressed in Drosophila S2 cells.
Distributions of background-corrected fluorescence of (A) kinesin-1-EGFP expressed in Drosophila melanogaster S2 cells, (B) DHC-EGFP, and (C) p50-EGFP. The dashed line in each histogram denotes the mean value of the kinesin-1-EGFP dataset. (D) Box and whisker plots of the kinesin-1, DHC, and p50 fluorescence intensities (kinesin-1, n = 100 puncta; DHC, n = 71 puncta; p50, n = 38 puncta). The reported p-values were determined by a randomization method: n.s. is not significant (p > 0.05).
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Figure 3—figure supplement 2—source data 1
Excel spreadsheet containing the underlying data and numerical values for plots in Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/94963/elife-94963-fig3-figsupp2-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene [Homo sapiens (Human)] | Dynein cytoplasmic 1 heavy chain 1 (DYNC1H1) | HGNC:HGNC:2961 | Gene ID: 1778 | https://www.ncbi.nlm.nih.gov/gene/1778 |
| Gene [Homo sapiens (Human)] | Dynactin subunit 2 (DCTN2) | HGNC:HGNC:2712 | Gene ID: 10540 | https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=DetailsSearch&Term=10540 |
| Cell line (Human) | Parental HeLa | American Type Culture Collection (ATCC) | ATCC: CCL-2 | |
| Cell line (Human) | DHC (FKBP-EGFP-KI/+) KI = Knock-in | This study | HeLa with tags added to the C-terminus of DYNC1H1/DHC | |
| Cell line (Human) | p50(FKBP-EGFP-KI/+) KI = Knock-in | This study | HeLa with tags added to the C-terminus of DCTN2/p50 | |
| Cell line (Drosophila melanogaster) | Drosophila Schneider 2 (S2) cell line | American Type Culture Collection (ATCC) | ATCC: CRL-1963 | Also referred to as SL2 or DMel2 |
| Recombinant DNA reagent | pMT-Kinesin-1-EGFP | This study | Modified from original construct in Ye et al., 2018 | |
| Antibody | Anti-dynactin p50 (mouse polyclonal) | BD Transduction Laboratories (Cat. # 611002) | 1:1000 dilution used for western blots | |
| Antibody | Donkey-anti-mouse IgG secondary antibodies conjugated with HRP | Jackson ImmunoResearch Laboratories, Inc (Code: 715-035-151) | RRID:AB_2340771 | 1:5000 dilution used for western blots |
| Recombinant DNA reagent | pB80-hsKIF5B(1-560)-L-GFP | Addgene plasmid # 193716 | RRID:Addgene_193716 | |
| Recombinant DNA reagent | pSpCas9(BB)-2A-Puro (PX459) | Addgene plasmid # 62988 | RRID:Addgene_62988 | |
| Chemical compound, drug | SiR-Tubulin | Cytoskeleton | Cat. #CY-SC002 | |
| Chemical compound, drug | Antibiotic/antimycotic cocktail | Sigma-Aldrich | A5955-100ML | |
| Software, algorithm | MetaMorph | MetaMorph | ||
| Software, algorithm | ImajeJ,Fiji | NIH | ||
| Software, algorithm | MS office | Microsoft | ||
| Software, algorithm | Prism | GraphPad | ||
| Software, algorithm | R | https://www.R-project.org/ | ||
| Software, algorithm | PlotsOfDifferences | https://huygens.science.uva.nl/PlotsOfDifferences | Goedhart, 2019 |
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Human dynein–dynactin is a fast processive motor in living cells
eLife 13:RP94963.
https://doi.org/10.7554/eLife.94963.3
